MF Researcher Database

Researchers Database

Advance

OHYA Susumu

    Graduate School of Medical Sciences Department of Pharmacology　Professor
Last Updated :2024/03/19

Researcher Information

Degree

  • Ph.D.（Nagoya City University）

URL

ORCID ID

J-Global ID

Research Interests

  • イオンチャネル   病態分子薬理学   Pharmacology   

Research Areas

  • Life sciences / Pharmacology

Academic & Professional Experience

  • 2017/09 - Today  Nagoya City UniversityGraduate School of Medical SciencesProfessor
  • 2012/04 - 2017/08  Kyoto Pharmacutical UniversityFaculty of Pharmaceutical SciencesProfessor
  • 2005/04 - 2012/03  Nagoya City UniversityGraduate School of Pharmaceutical SciencesAssociated Professor
  • 1995/04 - 2005/03  Nagoya City UniversityGraduate School of Pharmaceutical SciencesAsistant Professor

Education

  • 1994/04 - 1995/03  Nagoya City University  大学院薬学研究科  博士後期課程
  • 1992/04 - 1994/03  Nagoya City University  Graduate School of Pharmaceutical Sciences  博士前期課程
  • 1988/04 - 1992/03  Nagoya City University  Faculty of Pharmaceutical Sciences  製薬学科

Association Memberships

  • THE JAPANESE SOCIETY FOR IMMUNOLOGY   THE JAPANESE CANCER ASSOCIATION   American Physiological Society   日本生理学会   日本薬剤師会   Physiological Society   Biophysical Society   日本薬学会   日本薬理学会   

Published Papers

Books etc

  • eReview薬理学
    (Joint translation)Elsevier Japan KK 2020
  • 医療薬学III
    大矢 進 (Contributor感覚器・皮膚の疾患に用いられる代表的な薬物の基本構造と薬効（薬理・薬物動態）)東京化学同人 2017/04
  • Pharmacology
    OHYA Susumu (Contributor自律神経系に作用する薬物)廣川書店 2015/08

MISC

Awards & Honors

  • 2006 日本薬学会奨励賞（平成18年度）
     JPN
  • 2005 最優秀ポスター発表賞（US-Jpan Conference on Drug Development & Rational Drug Design)

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2026/03 
    Author : 松井 未来; 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2020/04 -2024/03 
    Author : 大矢 進; 鬼頭 宏彰
     
    本研究の目的は、三次元（3D）スフェロイド培養システムを用いてin vitroで再現した腫瘍微小環境でのがん幹細胞能および抗がん剤耐性能の獲得におけるカルシウム活性化カリウムチャネル（KCaチャネル）の病態生理学的意義を解明し、KCaチャネル作用薬の悪性がん治療薬としての潜在性を示すことである。本年度の研究計実施計画では、ヒト前立腺がん細胞における①抗アンドロゲン剤耐性獲得のメカニズムを解明するとともに、②KCa1.1阻害薬による抗アンドロゲン剤耐性克服効果を検討した。本研究では、アンドロゲン依存性ヒト前立腺がん細胞LNCaPを用いて以下のことを明らかにした。(1) スフェロイド培養によりLNCaPのKCa1.1活性が亢進しており、ユビキチンE3リガーゼFBXW7の発現抑制によるKCa1.1のタンパク分解の抑制が関与することを明らかにした。(2) LNCaPスフェロイド培養モデルにおいて、KCa1.1阻害薬の前投与によりdoxorubicin耐性が克服された。また、doxorubicin耐性獲得とKCa1.1阻害によるその克服には、ABCトランスポーターMRP5が関与することが示唆された。(3) LNCaPスフェロイド培養モデルにおいて、抗アンドロゲン剤耐性獲得にユビキチンE3リガーゼMDM2を介したアンドロゲン受容体ARタンパク分解促進が関与しており、KCa1.1阻害薬の処置によりMDM2発現が抑制されることにより、抗アンドロゲン剤耐性が克服された。さらに、「乳がん患者の腫瘍サンプルを用いたイオンチャネル発現の網羅的解析」を実施した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/10 -2023/03 
    Author : 今泉 祐治; 大矢 進; 山村 寿男; 鈴木 良明; 鬼頭 宏彰
     
    １，IL-10/IL-17Aを産生する制御性T細胞は、炎症性腸疾患（IBD）のような自己免疫疾患の発症・増悪の抑制に関与することが知られている。我々はIBDモデルマウスやin vitroで誘導した制御性T細胞において、Ca2+活性化K+チャネル（KCa3.1）阻害薬がBlimp1、E4BP4、KLF4といったIL-10/IL-17Aの転写制御因子の発現が亢進することで、IL-10/IL-17A発現を亢進させることを明らかにした。(Ohya et al., 2021)。さらに、JNK阻害薬がKCa3.1阻害によるIL-10発現上昇を抑制することと、KCa3.1阻害薬がリン酸化JNKおよびc-Junの発現を上昇させることを見出した。これにより、JNK/c-Junシグナルが、制御性T細胞におけるKCa3.1阻害誘導性のIL-10発現亢進に関与することを明らかにした (Matsui et al, 2022)。 ２，敗血症では、炎症反応による骨芽細胞障害を介したIL-7産生抑制が免疫細胞数の減少を引き起こす。我々は、マウス骨芽細胞株MC3T3-E1の骨芽細胞分化に内向き整流性K+チャネルKir2.1の機能亢進が重要な役割を果たすことを明らかにした。さらに、miR-106p-5pの発現減少がKir2.1の発現上昇に寄与することが明らかとなった。現在、慢性炎症時におけるKir2.1やmiRNAの発現活性変化と骨芽細胞機能との関連について検討中である。 ３，血管に対する持続的なストレス負荷が興奮転写連関を介してケモカインなど白血球集積を引き起こす分子の遺伝子発現の転写を引き起こすことを明らかにした。これにより、マクロファージが血管壁に集積して慢性炎症が引き起こされることで、血管リモデリングが引き起こされることを見出した（Suzuki et al, 2022）。 ４，門脈圧亢進症モデルマウスの門脈平滑筋細胞では、アンジオテンシンⅡのはたらきによりTMEM16Aの発現が減少して、自発運動に異常が生じることを明らかにした（Kondo et al, 2022)
  • カリウムチャネルを分子標的とした炎症性腸疾患に関する研究
    ブリストル・マイヤーズ スクイブ：腫瘍免疫・免疫・循環疾患領域 研究助成
    Date (from‐to) : 2019/10 -2021/03 
    Author : 大矢進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : Fujii Masanori
     
    Itch is the most bothersome symptom of atopic dermatitis, a common chronic skin disease. Mas-related G protein-coupled receptors (Mrgpr) are interesting therapeutic targets for intractable itch because they are reported to mediate non-histminergic itch; however, the role of Mrgpr in itch in atopic dermatitis remains unknown. In this study, we examined whether Mrgpr are involved in itch responses in mice suffering from atopic dermatitis. The major findings of the present study are as follows: 1) Ablation of MrgprA3-expressing sensory neurons by toxin receptor-mediated cell knockout suppressed mechanically induced scratching in atopic dermatitis mice. 2) Tacrolimus, a therapeutic agent for atopic dermatitis, may act directly on dorsal root ganglion cells expressing MrgprA3 to suppress their neural activation, leading to inhibition of itch sensation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2016/04 -2019/03 
    Author : OHYA Susumu; MURAKI Katsuhiko
     
    Upregulated K2P5.1 and KCa3.1 K+ channels are implicated in the pathogenesis of inflammatory bowel disease (IBD). Dysregulated K2P5.1 splicing by the pre-mRNA splicing inhibitor in activated CD4+ T cells disappeared K2P5.1 activity in CD4+ T cells. It may be effective against K2P5.1-related autoimmune diseases. Histone deacetylase (HDAC) inhibitors selective for class I HDAC, HDAC2 and HDAC3 suppressed the upregulation of KCa3.1 and its increased activity in CD4+ T cells of IBD model mice, suggesting that epigenetic modification of KCa3.1 contributes to enhanced inflammatory cytokine production and T cell activation. Interleukin-10 (IL-10) facilitating escape from cancer immune surveillance is negatively regulated by KCa3.1 activation by blocking the nuclear accumulation of phosphorylated Smad2 through calmodulin kinase II signaling suggesting that KCa3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10.
  • カリウムチャネル阻害による抗炎症性サイトカインIL-10産生増大機構の解明
    ソルト・サイエンス研究財団：平成29年度助成
    Date (from‐to) : 2017/04 -2018/03 
    Author : 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2016/03 
    Author : Niwa Satomi; NAIKI Taku; SASAKI Shoichi; TAKAHASHI Satoru; OHYA Susumu
     
    Ca2+-activated K+ channels (KCa) are key molecules in cancer progression and are considered to be potential targets for cancer therapy. KCa2.2 was predominantly expressed in human prostate cancer (PCa) tissues and human PCa cell lines, LNCaP and VCaP, with higher expression levels of androgen receptor (AR). A Ca2+-activated K+ channel blocker, UCL1684 suppressed the cell proliferation through the inhibition of the store-operated Ca2+ entry (SOCE) in LNCaP cells. The anti-androgenic agents and the siRNA-mediated inhibition of AR expression downregulated the expression levels of KCa2.2 in LNCaP cells. Additionally, the expression levels of KCa2.2 was upregulated with the upregulation of AR transcripts under long-term, androgen-deficient condition, whereas it was downregulated under short-term condition. These results suggest that KCa2.2 might induce a possible candidate for novel treatment target of Castration-Resistant Prostate Cancer, CRPC.
  • Tリンパ球におけるtwo-pore型K+チャネルK2P5.1の生理的役割とその新規阻害機構の解明
    ソルト・サイエンス研究財団：平成27年度助成
    Date (from‐to) : 2015/04 -2016/03 
    Author : 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2013/04 -2016/03 
    Author : Ohya Susumu; FUJII Masanori
     
    The two-pore domain K+ (K2P) channels are possible therapeutic targets for autoimmune diseases and several cancers. We elucidate the pathological significance of the K2P5.1 K+ channel in inflammatory bowel disease (IBD). Significant upregulation of K2P5.1 K+ channel were observed in the CD4+ T cells of the IBD model. The knockout of K2P5.1 in mice significantly suppressed the disease severity in the IBD model. Additionally, we identified an N-terminus-lacking, novel splicing isoform of K2P5.1 K+ channel, K2P5.1B from the human lymphoid tissues. In a heterologous expression system, K2P5.1B inhibited the plasma membrane trafficking of K2P5.1A. The pre-mRNA splicing inhibitor significantly enhanced the expression levels of K2P5.1B in human leukemic K562 cells, resulting in decrease in the K2P5.1 activity. The pre-mRNA splicing mechanism underlying the posttranscriptional regulation of K2P5.1 K+ channel may be a new therapeutic strategy for autoimmune diseases and cancers.
  • カリウムチャネル新規阻害機構の解明
    上原記念生命科学財団：平成24年度研究推進特別奨励金
    Date (from‐to) : 2014/04 -2015/03 
    Author : 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/11 -2014/03 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; HIGUCHI Tsunehiko; ASAI Kiyofumi; HIRONO Syuichi
     
    The present study revealed that, in non-excitable cells such as endothelial cells, lymphocytes, chondrocytes and airway cilliated cell, Ca2+-activated K+ (KCa) channels and store-operated Ca2+ (SOC) channels play significant roles in the positive feedback mechanism for the regulation of intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i elevation elicites cellular responses to various types of stimula under physiological conditions or is involved in pathological processes. In brain capillary endothelial cells and chondrocytes, Ca2+-release activated Ca2+ channel is a major component of SOC channels and regulates cell proliferation. In T lymphocytes isolated from inflammatory disease model mice, the change in expression level of intermediate-conductance KCa channel is related to pathological processes. In ciliated cells, ATP-sensitive K+ channel contribute to the positive feedback mechanism for [Ca2+]i, and facilitate ciliary movement and consequently promote airway clearance.
  • ストレス応答性調節におけるリンパ球Ca2+活性化K+チャネルKCa3.1の役割
    薬理研究会：平成24年度研究助成
    Date (from‐to) : 2012/04 -2013/03 
    Author : 大矢 進
  • 免疫シナプスにおけるTwo-pore domain K+チャネル輸送機構の解明
    持田記念医学薬学振興財団：第30回（平成24年度）研究助成金
    Date (from‐to) : 2012/04 -2013/03 
    Author : 大矢 進
  • カルシウム活性化カリウムチャネルの新規発現調節機構とその創薬への応用
    武田科学研究振興財団：2011年度 薬学系研究奨励継続助成
    Date (from‐to) : 2011/04 -2013/03 
    Author : 大矢 進
  • 炎症性疾患におけるTリンパ球カリウムチャネル活性・発現調節
    鈴木謙三記念医科学応用研究財団：平成23年度調査研究助成金
    Date (from‐to) : 2011/04 -2012/03 
    Author : 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2012 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Toshio
     
    To provide a simple but high throughput screening method for compounds acting on ion channels, a new recombinant cell line, in which single action potential (AP) induced cell death, was produced by gene transfection. Mutated human cardiac Na^+channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild type inward rectifier K^+channel, Kir2.1 were stably co-expressed in HEK293 cells (IFM/Q3+K_2.1). In IFM/Q3+K_2.1, application of single electrical stimulation (ES) elicited a long AP lasting over 30 s and led cells to die by over 70 %, while HEK293 co-transfected with wild type Nav1.5 and K_2.1 fully survived. The additional expression of hERG K+channels in IFM/Q3+K_2.1 shortened the duration of evoked AP and, thereby, markedly reduced the cell death. The treatment of the cells with nifekalant, E-4031, cisapride, terfenadine and verapamil, hERG channel inhibitors, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. Results indicate the high utility of this cell system for hERG K+channel safety assay. Moreover, to develop a screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing IMF/Q3, K_2.1 and Kv1.3 or Kv1.5 were introduced as IFM/Q3+K_+Kv1.3 and IFM/Q3+K_+Kv1.5, respectively. Co-expression of Kv1.3 or Kv1.5 to IFM/Q3+K_ shortened the evoked APs and prevented the cell death. In the presence of margatoxin, a selective Kv1.3 blocker, ES induced the cell death in IFM/Q3+K_+Kv1.3, but not in IFM/Q3+K_+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5 blocker, and citalopram, a 5-HT uptake-inhibitor, in IFM/Q3+K_+Kv1.3 were almost identical to those in IFM/Q3+K_+Kv1.5. It was, thereby, found that acacetin and citalopram block both Kv1.3 and Kv1.5 without significant selectivity. The new cell lines for hERG, Kv1.3 or Kv1.5 channel inhibition assay fit to the high-throughput screening because of its simplicity, accuracy and high cost-performance.
  • カルシウム活性化カリウムチャネルの新規発現調節機構とその創薬への応用
    武田科学研究振興財団：2009年度 薬学系研究奨励助成
    Date (from‐to) : 2009/04 -2011/03 
    Author : 大矢 進
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2009 -2011 
    Author : OHYA Susumu
     
    The novel spliced variant of intermediate-conductance Ca^<2+>-activated K^+channel K_ 3.1(K_ 3.1-sp/K_ 3.1b) has been identified from the human immune tissues, which were lacking the N-terminal domains of the original K_ 3.1a as a result of alternative splicing. The cellular distribution of CFP-tagged K_ 3.1a and/or YFP-tagged K_ 3.1-sp isoforms showed that K_ 3.1-sp suppressed the localization of K_ 3.1a to the plasma membrane, and co-expression of K_ 3.1-sp with K_ 3.1a suppressed IK_ channel activity of K_ 3.1a in a dominant-negative manner. In addition, the up-regulation and over-expression of K_ 3.1-sp suppressed thymocyte growth by down-regulation of IL-2 transcripts. These suggest that the N-terminal domain of K_ 3.1 is critical for channel trafficking to the plasma membrane, and that the fine tuning of IK_ channel activity modulated through alternative splicing may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; ASAI Kiyofumi; HIGUCHI Tunehiko
     
    The present study revealed that functional expression of Ca^<2+>-activated K+ (BK, IK, SK) channels in non-excitable cells, such as chondrocytes, vascular endothelial cells and T-lymphocytes, substantially contributes to sustained increase in intracellular Ca^<2+> concentration in response to various types of stimuli. We proved that Ca^<2+>-activated K+ channels play the central roles in the positive feedback mechanism for the regulation of intracellular Ca^<2+> concentration via the membrane hyperpolarization, which increases the driving force of store-operated Ca^<2+> influx through non-selective cation channels.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2008 -2009 
    Author : 今泉 祐治; 大矢 進; 山村 寿男
     
    細胞膜上でイオンチャネルは他分子と特定の分子複合体(トランスポートソーム)を形成して、初めて正常な生体内機能を果たしている場合の多いことが明らかになりつつある。本研究は、平滑筋、およびペースメーカー細胞としての間質系カハール細胞、非興奮性細胞としての軟骨細胞におけるCa^<2+>活性化K^+チャネル(BKチャネルなど)とその他の細胞膜上のイオンチャネルやトランスポーターやカベオリン、更には小胞体膜上のリアノジン受容体との分子間連関の可能性とトランスポートソームの実体を一分子可視化法により明らかにすることを、目的としている。蛍光タンパクでラベルされたこれら分子の遺伝子を上記細胞に導入・発現させ、全反射顕微鏡で可視化するとともに、その機能を電気生理学的に解析した。 (1) リアノジン受容体とBKチャネルの機能連関を可視化するとともに、男性ホルモンによる発現調節機構を明らかにした(J Pharmacol Sci, 2009)。 (2) 軟骨細胞由来の培養細胞において、細胞内Ca2+濃度制御機構において、非選択的陽イオンチャネルとの機能連関により、BKチャネルなどのCa^<2+>活性化K^+チャネルが重要な役割を果たしていることを明らかにした(Am J Physiol, 2010)。またCl^-チャネルとの機能連関を明らかにした(J Pharmacol Sci, 2010) (3) Na^+-Ca^<2+>交換体を高発現したマウス膀胱平滑筋において、その生理機能を明らかにした。(J Pharmacol Sci, 2010)
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2008 
    Author : KIMURA Kazunori; OHYA Susumu
     
    我々はラットの動脈性ED(erectile dysfunction)モデルを用いて、vardenafil慢性投与によるED改善メカニズムを血流動態学および組織学面から検討した。勃起機能の評価は海綿体神経刺激による海綿体内圧変化を調べた。その結果、vardenafil投与によって陰茎海綿体への側副血行路の発達と海綿体平滑筋の構造保持が確認され、動脈性EDラットの勃起機能が改善された。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2006 -2007 
    Author : 大矢 進
     
    本研究の目的は,(1)Ca^<2+>活性化K^+チャネルを介した神経,心筋細胞保護作用の分子機構,(2)Ca^<2+>活性化K^+チャネル開口薬による神経,心筋細胞保護を明らかにすることであった。(1)については,18年度の研究成果を発展させ,Ca^<2+>活性化K^+チャネルの活性化により惹起される細胞内Ca^<2+>流入経路の分子基盤の同定に成功し,「Ca^<2+>活性化K^+チャネルのMAKキナーゼ経路,カスパーゼ経路への影響」についても研究成果が得られた(Yamazaki, et. al.,2007)。また,松果体細胞におけるCa^<2+>活性化K^+チャネルのCa^<2+>オシレーション発生への寄与について,Ca^<2+>イメージング解析により明らかにした(第111回日本薬理学会近畿部会にて発表)。最近では,扁桃体神経細胞においてアンドロゲン枯渇によりCa^<2+>活性化K^+チャネル発現,活性が顕著に減少することを明らかにした(第85回日本生理学会大会にて発表)。(2)については,Ca^<2+>活性化K^+チャネルβ1サブユニットの変異体を作成し,β1サブユニット選択的新規Ca^<2+>活性化K^+チャネル開口薬DiBAC4(3)の認識部位に第2膜貫通ドメインのいくつかのアミノ酸が関与することを明らかにした(日本薬学会第128年会にて発表)。また,Ca^<2+>活性化K^+チャネル開口薬ジクロロデヒドロアビエチン酸がミトコンドリア呼吸,容積,Ca^<2+>過負荷を改善することを明らかにした(投稿中)。ミトコンドリアCa^<2+>活性化K^+チャネルの分子実体については,候補分子の同定を含めて一定の研究成果が得られたものの,再構築系での更なる研究成果の蓄積が必要である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2006 -2007 
    Author : 今泉 祐治; 大矢 進; 山村 寿男; 村木 克彦
     
    <細胞内CAa^<2+>信号の電気信号への変換機構におけるトランスポートソーム機能と構造実体の解析>2型リアノジン受容体(RyR2)異型接合性欠損マウス膀胱平滑筋を用いてRyR2の寄与を明らかにした。膀胱平滑筋においてRyR2を介した自発Ca^<2+>遊離(Ca^<2+>spark)の発生とそのCa^<2+>信号をSTOCsという電気信号に変換するトランスポートソーム機能は,尿貯留・排泄調節という膀胱機能発現において根源的な果たす役割を果たすことが示され,当該トランスポートソームの実体はカベオラ構造内に存在することが強く示唆された。さらにこのような信号機構がカハール間質細胞において生じ,ペースメーカー電位発生の根源となっている可能性を再構築系を用いて明らかにした。 <大コンダクタンスCa^<2+>活性化K^+(BK)チャネルのβサブユニット特異的開口物質の発見>電位感受性蛍光色素のDiBAC_4(3)および関連オキソノール色素にBKチャネルβ1およびβ4サブユニット選択性(β2には無効)を有するBKチャネル開口作用があることを発見し,創薬の可能性を示した。 <一分子可視化によるCa^<2+>信号から電気信号への変換トランスポートソームの機能解析>トランスポートソームにおけるCa^<2+>信号から電気信号への変換に関する分子機構解明における新たな手法として一分子レベルでの可視化技術を導入した。全反射蛍光顕微鏡とホールセルクランプ法の併用により,電位固定化で細胞膜直下200nm以内でのナノスケールの蛍光分子動態が測定可能となった。また記録電極からCa^<2+>蛍光色素Fluo4を細胞内に導入し,脱分極刺激時の膜直下の局所Ca^<2+>濃度変化をナノスケールで計測することが可能となった。この方法を用いて電位固定化でのチャネルを中心としたトランスポートソーム機能の定量的ナノイメージング解析を行った。トランスポートソームの信号変換分子機構を解明する上で一分子可視化技術は画期的な技術となる可能性を示した.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2007 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; TOGARI Akihumi
     
    In excitable cells, such as CNS neurons, negative feedback regulation systems for intracellular Ca^<2+> homeostasis work to prevent Ca^<2+> overlord, when excess excitability and resulting excess Ca^<2+> influx occurs. Activation of Ca^<2+> activated K+ (Kca) channels is know to be one ` of the most important components responsible for the negative feedback regulation of [Ca2^+]_i in excitable cells. This project was undertaken to elucidate the molecular mechanism for the regulation of Kca channel activity and to search changes in the regulation diseases. The goal of this project is to find out molecular seeds targeting on K_ca channels in some diseases. The following development was obtained during the research period. (1) It was found that small conductance K_ca (SK) channel in vascular endothelial cell lines originally derived from bovine blood-brain barrier has significant functional role in endothelial; proliferation stimulated by ATP presumably released from astrocytes in CNS. (JBC,2006; J Pharmacol Sci, 104, 2007). (2) The deep impact of ryanodine receptor type2 (RyR2) to the mechanism for negative feedback regulation of [Ca^<2+>], via spontaneous Ca^<2+> release(Ca^<2+> spark) from sarcoplasmic reticulum and subsequent activation of large conductance Kca (BK) channel was found using urinary bladder smooth muscle cells from wild type and RyR2 heterozygous KO mice. A line of evidence indicates that RyR2 contributes to the bladder continent as 'a key molecule regulating resting membrane potential and muscular tonus as well as excitation-contraction coupling (J Physiol, 2007, J Pharmacol Sci, 103, 2007). (3) It was found that potential sensitive oxonol dyes act as potent BK channel openers. It is the first synthesized compound which shows opening property selective to BK81 and 84 subunits over BK62. The oxonol compounds may be a seed of 8 subunit selective BK channel opener (Mol Pharmacol, 2007). (4) It has been known that the contractile responses of isolated large arteries from spontaneously hypertensive rats (SHR) to agonists are markedly potentiated in low pH bathing solution. It was found that the enhanced expression of BK channels in arterial smooth muscles of SHR and its sensitivity to extracellular pH are responsible for the acid pH induced potentiation of contraction (Am J Physiol, 2007).
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2005 -2006 
    Author : 今泉 祐治; 大矢 進; 山村 寿男
     
    全反射蛍光顕微鏡を利用して、生きた細胞内で機能している細胞内小器官、特に小胞体の膜上に存在する特定の蛋白質(特にはリアノジンCa^<2+>遊離チャネル)のリアルタイムでの機能を一分子可視化法により、解明することを試みた。さらにその技術を一般化し、その他の小胞体上の蛋白質、あるいはその他のオルガネラ(ミトコンドリアなど)の膜表面蛋白を一分子可視化し、その機能解析に新分野を切り開くための端緒とするべく検討した。 (1)膜電位固定下で平滑筋細胞膜上の1分子のまたは凝集した分子群のCa^<2+>チャネルが脱分極で開口することにより、細胞膜直下の筋小胞体からのリアノジン受容体を介したCa^<2+>遊離を可視化することに成功した(07年日本薬理学会年会発表;論文投稿準備中)。 (2)YFPでラベルされた2および3型リアノジン受容体をHEK293細胞に発現させ、リアノジン受容体開口による自発的Ca^<2+>遊離現象を一分子可視化することを試行している。 (3)CFPラベルされた大コンダクタンスCa^<2+>活性化K^+チャネルをHEK293細胞に発現させ、機能的クラスター形成の過程を一分子可視化法により明らかにした。また(2)と同時に発現させることにより両分子の機能連関の可視化を検討している。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2004 -2005 
    Author : 大矢 進
     
    本年度の研究実施計画では、「K^+チャネル電位依存性制御の分子機構解明」と「電位依存性K^+チャネル開口薬の探索」に主眼をおき、次の検討事項を掲げた。 1.高効率薬物探索系における電位依存性K^+チャネル開口薬の探索 2.電位センサー変異体による開口薬作用部位の同定 1については、研究代表者が樹立した電位依存性Ca^<2+>活性化K^+チャネル定常発現細胞株を用いて開口薬の候補化合物を探索した。電気生理学的手技により解析した結果、脳内化学物質であるアナンダミドや樹脂酸誘導体がK^+チャネル開口作用を有することを明らかにすることができた(研究業績参照)。また、これまで分子実体が不明であったミトコンドリアCa^<2+>活性化K^+チャネルの分子実体の一部(mitoK_-β1)を明らかにし、ミトコンドリア内膜においてmitoK_-β1とシトクロムc酸化酵素が複合体を形成する可能性を示した(研究業績参照)。本研究では、ミトコンドリア膜電位、ミトコンドリア呼吸の可視化解析や実験的虚血心筋細胞モデルによりK^+チャネル開口薬による心筋細胞保護作用の機構の一部も明らかにした。 2については、遺伝子改変技術により電位依存性K^+チャネル、Kv1.1チャネルの点変異体を複数作成し、樹脂酸誘導体によるK^+チャネル開口作用や電位依存性制御の分子機構ついて検討した。その結果、樹脂酸誘導体による電位依存性制御に関与する部位をある程度同定することができた(学会発表済、投稿中)。 最近では消化管間質細胞腫におけるK^+チャネル遺伝子発現解析を行い、電位依存性K^+チャネルが腫瘍増殖に関与している可能性を示した(研究業績参照)。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2003 -2004 
    Author : 今泉 祐治; 大矢 進
     
    現在、汎用されている代表的なオキソノール系蛍光色素としてDiBAC_4(n)やDisBAC_4(n)などが挙げられる。陰イオンであるDiBACは細胞膜を良く透過するため、細胞外液中に存在させると膜電位差が減少した時には細胞内へより多く分布し、不特定の細胞質蛋白と結合して、細胞内からの蛍光強度を増す。細胞外のfreeのDiBACは微弱な蛍光しか発しない。アーチファクトが生じやすい理由は、本来は極めて短い色素の蛍光寿命が、不特定の細胞質蛋白との結合により延長されて測定が可能となっているという根本的な測定原理に由来する。蛋白と色素の結合に影響を与える化合物は、イオンチャネルに作用しなくても蛍光強度を変化させるからである。上記の欠点を解消するため、次のような系を考案した。DiBAC系膜電位感受性蛍光色素に標識化学構造を化学合成により付加した。一方、その標識部位を特異的に認識する蛋白を遺伝子導入により細胞に高発現させることを試みた。付加的化学構造を持つ色素は特異的結合蛋白に優先的に結合するため、不特定の細胞内蛋白との結合は防がれ、かつより高効率の蛍光を発することが可能性となると考えた。構造が比較的単純な精巣型アンジオテンシン転換酵素タンパクの一部とその阻害薬カプトプリル類縁化合物の利用を検討した。特異的結合蛋白を低分子化することにより色素との結合・解離速度を上昇させることができると想定した。以上から、当方法によりアーチファクトの減少と反応速度の上昇が得られると推測し検討を加えたものの、現在のところ従来と比較して明らかな反応速度の変化は観察されていない。DiBACが既に細胞質のタンパクのうちでも比較的低分子なものに良く結合していた可能性、まだ低分子化が不足している可能性、細胞内では何らかの理由で結合能が低下しているなどの理由が考えられるのでさらに検討が必要である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2004 
    Author : IMAIZUMI Yuji; MURAKI Katsuhiko; OHYA Susumu; OHWADA Tomohiko
     
    Although the increase in intracellular Ca^<2+> concentration ([Ca^<2+>]i) is commonly observed in responses to various types of stimuli, the excess increase in [Ca^<2+>]i, or in other words, overload of cells with calcium is one of the key steps and very popular in the process of accumulation in cellular damages under pathophysiological settings. To minimize calcium overload, cells have various systems to extrude Ca^<2+> to and/or prevent Ca^<2+> entry from outside. The Ca^<2+> entry is usually due to opening of two separate types of Ca^<2+> entry channels; voltage-dependent Ca^<2+> channels (VDCCs) and non selective cation channels. Ion channels, whose activities are directly modulated by [Ca^<2+>]i, strongly contributes to the regulation of Ca^<2+> entry via the changes in membrane potential. Large conductance Ca^<2+> activated K^+ (BK) channels are ubiquitously expressed in excitable cells except cardiac myocytes and also expressed in some non-excitable cells. The activation of BK channel induces membrane hyperpolarization, reduces VDCC activity and minimizes Ca^<2+> overload in excitable cells. We surveyed low molecular natural products mainly from plants to find out new prototype of BK channel opener, since this type of agents may reduce the hyper contractility of smooth muscle tissues or Ca^<2+> over load in neurons under pathophysiological conditions. Among over 60 natural products and their synthesized derivatives, we found pimaric acid (PiMA)and related compounds as potent openers of BK channel. Effects of PiMA and other compounds on BK channels were examined using HEK293 cells, in which either the a-subunit of BK channel (HEKBKα) or both α and 01 (HEKBKαβ1) subunits was heterologously expressed. Effects of these compounds (10μM) on the membrane potential of HEKBKαβ1 were monitored by use of DiBAC_4(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BKαβ1 opening under whole cell voltage-clamp showed that these six compounds activated BKαβ1 in a very similar concentration range (1-10 μM), in contrast abietic acid, sclareol and methyl pimarate had no effect. PIMA did not affect the charybdotoxin-induced block of macroscopic BKaβ1 current. Single channel recordings of BKαβ1 in inside-out patches showed that 10 μM PiMA did not change channel conductance, but significantly increased its open probability due to increase in sensitivity to Ca^<2+> and voltage. Since co-expression of β1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKα subunit. PiMA was selective to BK over cloned small and intermediate Ca^<2+> activated K^+ channels. It can be concluded that PiMA (>1μM) increases Ca^<2+> and voltage-sensitivity of BKα when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BKα activation. Moreover, we found that BK channel-like K^+ channels, which may be expressed in mitochondria of cardiac myocytes, are also activated PiMA. The protective effects of PiMA to reduced cell injury in ischemic conditions were also detected in rat cardiac myocytes. Taken together, we found a useful compound, PiMA, as a prototype of BK channel opener and obtained basic information about the activity-structure relationships for BK channel opener.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2002 -2003 
    Author : 大矢 進
     
    本研究では、電位依存性カリウムチャネル機能制御分子のうち、心筋、神経においてQT延長症候群、癲癇に関与する電位依存性カリウムチャネル、ERG1-3、KCNQ1-5の機能制御分子、KCNE1-5と早期不活性化カリウムチャネルKv4.3の機能制御分子KChIp-4の平滑筋機能における役割について検討した。 門脈は自動収縮を惹起する組織であり、その機能調節には心筋と向様にK^+チャネルが重要な役割を果たしている。本研究では、マウス門脈平滑筋に発現する新規KCNQ1スプライスバリアント、KCNQ1bをクローニングし、その電気生理学的特性について検討した(「研究発表」参照)。その結果、心筋ではKCNE1がKCNQ1aの機能調節しているのに対して、門脈平滑筋ではKCNE3がKCNQ1bの機能調節を行っていることを見出した(「研究発表」参照)。心筋KCNQ1、KCNE1においてloss of functionを伴なう遺伝的変異が報告されている。本研究において、KCNQチャネルの特異的阻害薬、linopirdineにより門脈平滑筋における活動電位幅が顕著に延長したことから、門脈血行異常症にもKCNQ1bやKCNE3の遺伝的変異が関与する可能性がある。 本研究課題において、消化管平滑筋においてKChIPサブタイプのうち、KChIP1,KChIP3が発現することを前年度報告した。本研究では、Kv4.3のC末端細胞内領域にアミノ酸点変異体を導入することにより、Kv4チャネルの電位依存性に寄与する正電荷アミノ酸を見出し、KChIPはN末端と相互作用するだけでなく(An F. et al.,Nature,2002)、C末端とも機能的に相互作用することを見出した(「研究発表」参照)。最近、国際共同研究によりL(米国カリフォルニア大学サンディエゴ校、Wayne Giles教授)、KChIP欠損マウスを用いて平滑筋機能異常に関する研究を開始した。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2000 -2001 
    Author : 大矢 進
     
    電気生理学実験により子宮をはじめとする各種平滑筋の収縮調節機構に多くのイオンチャネルやトランスポーターが寄与することが明らかとなっている。また、K^+チャネル開口薬は抗高血圧薬、抗不整脈薬といった循環器疾患作用薬としての適用だけでなく、切迫早産治療薬、抗気管支喘息薬、尿失禁抑制薬としての開発が期待されている。本研究では各種平滑筋(子宮を含む)に発現する電位依存性K^+チャネルの分子同定、発現分布、機能的集積を検討した。また、新規K^+チャネル修飾蛋白を単離し、発現分布を検討した。 30種類以上単離されているK^+チャネルサブタイプのうちラット各種平滑筋において主に5種類[早期不活性化(Kv4.3)、遅延整流性(Kv1.2、1.5、2.1)、Ca^<2+>活性化(BKα)チャネル]が発現しており、膜興奮性により発現分布が異なっていた(研究発表参照)。また、輸精管のような興奮性の高い内臓平滑筋細胞ではBKチャネル蛋白が細胞膜に集積して機能的クラスターを形成していた(研究発表参照)。妊娠子宮平滑筋におけるK^+チャネル遺伝子のup/down-regulationやBKチャネル蛋白集積の変化に関しては現在進行中である。最近、早期不活性化K^+チャネルを機能修飾するK^+ Channel-Inteacting Protein(KChIP)が単離された。本研究では心筋に特異的に発現する新規KChIPを2種類単離するとともに、平滑筋に発現するKChIPサブタイプを同定した(投稿中)。KChIPは早期不活性化K^+電流密度を増大させるため妊娠後期や心筋梗塞時の早期不活性化K^+電流の減弱にはKChIPのdown-regulationが寄与する可能性がある。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1999 -2000 
    Author : 渡辺 稔; 大矢 進; 村木 克彦; 今泉 祐治
     
    単離平滑筋細胞に電位固定法を適用し、膜電流を記録するとともに、記録電極内からfluo-3を細胞に負荷しレーザー共焦点顕微鏡を用いて細胞内Ca^<2+>分布画像を同時に取得する手法を用いて、精管・膀胱・門脈等の興奮性の高い平滑筋細胞における興奮収縮連関の画像解析とCa^<2+>依存性K^+チャネル活性化の解析を行い、次の知見を得た。(1)細胞膜直下に局在する特定の筋小胞体(の一部分)は、活動電位などの脱分極時に、電位依存性Ca^<2+>チャネルを介したCa^<2+>流入によるCa^<2+>遊離機構の起点となり、数百ミリ秒持続するCa^<2+>ホットスポットを形成する。(2)膜直下筋小胞体からの局所遊離Ca^<2+>によるK^+チャネル活性化は、活動電位波形を制御し活動電位発生頻度を調節する。(3)この局所Ca^<2+>遊離は他の筋小胞体へCa^<2+>遊離を伝播した場合にのみ、収縮を誘発する可能性が高い。(4)このようなCa^<2+>ホットスポット形成という特定機能を持つ膜直下筋小胞体の数は、1細胞当り比較的少数(<20)と考えられる。 一方、保持電位-40mV付近での観察から、膜直下の特定の筋小胞体(数箇所)から、自発的な一過性局所Ca^<2+>遊離(Ca^<2+>スパーク)が繰り返し生じ、近傍の細胞膜上のCa^<2+>依存性K^+チャネル(10-100個)を活性化することにより自発性・一過性の外向き電流(STOC)が生じることが示唆されている。Ca^<2+>スパークとSTOCの持続時間は半値幅50ミリ秒程度で、その発生に外液Ca^<2+>の流入は直接必要ではなく、特定の筋小胞体からのリアノジン受容体を介する自発性Ca^<2+>遊離によると考えられる。今回、上記の活動電位発生初期に重要な働きをする特定の筋小胞体のさらに一部で、Ca^<2+>スパークがほぼ定期的に生じるていること、それがSTOCと完全に同期していることを画像解析と膜電流の同時記録により明らかにした。Ca^<2+>スパークは心筋や骨格筋において興奮収縮連関の最少ユニットとして解析されている。一方、横行小管と筋小胞体の発達が悪い平滑筋においては、細胞膜直下の筋小胞体で生じるCa^<2+>スパークが細胞膜上のCa^<2+>依存性イオンチャネルを活性化して静止膜電位や興奮性、さらに筋緊張度の調節に関与していることが明らかとなった。静止時にSTOCを活性化するCa^<2+>スパーク部位は、脱分極時にはより大きく増幅され持続するCa^<2+>ホットスポット部位となる。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -1999 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation were examined using RT-PCR, semi-quantitative PCR and skinned fiber techniques. It was found that microinjection of AF64A, a novel choline uptake inhibitor, into anterior eye chamber of the rat resulted in changes in iris sphincter smooth muscle similar to those elicited by surgical parasympathetic denervation by removal of ciliary gangrionectomy. The changes were characterized in vitro by reduced contractile response to nerve stimulation presumably via the decrease in acetylcholine (ACh) release from parasympathetic nerve endings and by enhanced contractile responses both specific to exogenously applied ACh and nonspecific to serotonin and high KィイD1+ィエD1. Semi-quantitative PCR analyses of muscarinic receptors in rat iris indicate that mRNAs of m2, m3 and m4 subtypes are expressed in iris and that, unexpectedly, the expression level of m4 is much higher than that of m2 and m3. The sequence homology of m2, m3 and m4 in rat iris to those reported in genome or cDNA in other tissues of the rat was over 99%, while several differences in amino acid sequence in intracellular loops were found. Following the injection of AF64A, mRNA levels of m2 and m3 increased and that of m4 decreased, respectively. The CaィイD12+ィエD1 sensitivity of the contractile machinery measured in skinned fiber was slightly but significantly increased by AF64A injection. The increase in CaィイD12+ィエD1 sensitivity was abolished by addition of H7, protein kinase C inhibitor. AF64A injection resulted in marked increase in the maximum contractile response of iris sphincter muscle. A slight increase in amount of actin and no change in those of myosin and calmodulin in iris can not explain the increase in the maximum contractile response. A novel mechanism distinctive from the changes in major contractile and related regulatory proteins may underlie the nonspecific supersensitivity induced by AF64A.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -1999 
    Author : MAIZUMI Yuji; OHYA Susumu
     
    In situ hybridization and RT-PCR analyses has revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA of Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have divergent C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f are expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -1999 
    Author : IMAIZUMI Yuji; OHYA Susumu; MURAKI Katsuhiko; WATANABE Minoru
     
    The interrelationship between the ionic current density of two channels (voltage-dependent L-type CaィイD12+ィエD1 channels (VDCCs) and large conductance CaィイD12+ィエD1 activated KィイD1+ィエD1 channels (BKCs) and the mRNA expression level of their channel subtypes (αィイD21cィエD2 subunit of L-type CaィイD12+ィエD1 channels (α1C) and α/β subunits of BK channel (αBK/βBK)) was determined in rabbit smooth muscles. BK currents (IィイD2K-CaィエD2) in the rabbit aorta were much smaller than those in the rabbit vas deferens. However, when CaィイD12+ィエD1 current (IィイD2CaィエD2) was increased by Bay K 8644, IィイD2K-CaィエD2 was also markedly increased especially in aortic smooth muscle cells. The amount of mRNA contents of encoding α1C was apparently higher in urinary bladder and vas deferens than that in aorta and trachea. In contrast, the mRNA contents of encoding both αBK and βBK were observed at similar levels in their four tissues. These observation raised the possibility that quantitative differences in the VDCC and BKC may provide an explanation for the differences in membrane excitability between aorta and trachea versus urinary bladder and vas deferens. The effects of ruthenium red (RuR) on contractility were examined in skinned fibers of guinea pig smooth muscles. Contractions of skinned fibers of the urinary bladder were enhanced by RuR (ECィイD250ィエD2=60μM). The contraction at pCa 6.0 was increased to 320% of control by 100μM RuR. Qualitatively the same results were obtained in the ileal longitudinal smooth muscle layer and mesenteric artery. Maximal contraction induced by pCa 4.5 was not affected significantly by RuR. Application of microcystin, a potent protein phosphatase inhibitor, induced a tonic contraction of skinned smooth muscle at low [CaィイD12+ィエD1] (pCa >8.0). RuR had a dual effect on the microcystin-induced contraction : enhancement at low concentrations and suppression at high concentrations. The relaxation following the decrease in [CaィイD12+ィエD1] from pCa 5.0 to >8.0 was significantly slowed down by an addition of RuR. Phosphorylation of myosin light chain at pCa 6.3 was significantly increased by RuR. These results indicate that RuR markedly increases CaィイD12+ィエD1 sensitivity of the contractile system at least in part via inhibition of myosin light chain phosphatase. Spatiotemporal relationships between CaィイD12+ィエD1 sparks and the activation of transient ClィイD1-ィエD1 current were analyzed in rabbit atrial myocytes based on simultaneous measurements of two dimensional CaィイD12+ィエD1 images by fast scanning confocal microscopy and membrane currents under whole cell voltage-clamp. The results suggest that CaィイD12+ィエD1 sparks initiate some patterns of global [CaィイD12+ィエD1]ィイD2iィエD2, and that either a CaィイD12+ィエD1 hot spot or a CaィイD12+ィエD1 wave is the spatiotemporal summation of individual CaィイD12+ィエD1 sparks, which results in the activation of CaィイD12+ィエD1 dependent ion channels on the sarcolemma. It can be strongly suggested that a CaィイD12+ィエD1 spark is a functional unit not only in excitation-contraction (E-C) coupling but also in the activation of CaィイD12+ィエD1 dependent membrane current, mainly IィイD2Cl-CaィエD2, in rabbit atrial myocytes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1997 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The amount of mRNAs of muscarinic acetylcholine receptor subtypes, m1, m2, m3, m4 and m5, in the rat iris was determined quasi-quantitatively using RT-PCR methods. The mRNA expression levels of m2 and m3 were higher than those of m1 and m5 in the rat iris as in other smooth muscle tissues. It was characteristic in the rat iris that the m4 mRNA expression level was high. In addition, the cloning of cDNAs encoding m2, m3 and m4 subtypes in the rat iris was performed using RT-PCR methods. Amino acids sequence of m2 subtype in the rat iris included nine differences in comparison with that in the heart, whereas it was 100% identical to genomic one. To examine the mechanisms underlying the parasympathectomy-induced effects in the rat iris smooth muscles, the mRNA expression levels of muscarinic receptor subtypes were determined in iris from ciliaryganglionectomized rats. The results suggested that the parasympathectomy does not markedly affect the mRNA levels. It was also clarified that the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in the rat iris dilator under the resting conditions is higher than those in the iris sphincter and many other smooth muscles which do not have an inherent tone. Since the muscarinic receptor stimulation resulted in the decrease in [Ca^<2+>]_i in the rat iris dilator, the muscarinic relaxation appeared to be due to the decrease in [Ca^<2+>]_i. In denervated iris dilators, muscarinic receptor stimulation induced neither the decrease in [Ca^<2+>]_i nor the relaxation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1997 
    Author : WATANABE Minoru; OHYA Susumu; MURAKI Katsuhiko; IMAIZUMI Yuji
     
    Early inactivating K^+ (A type) current can be recorded widely in neuron, cardiac myocytes and smooth muscle cells. the current plays significant roles for the regulation of action potential firing and formation of the repolarizing phase of an action potential. Although several distinct genes encoding the K^+ channels responsible for A type K^+ current (I_A) in cardiac myocytes and brain have been reported, nothing is known in smooth muscle cells. The electrophysiological characteristics of I_A in smooth muscle cells (vas deferens, colon and ureter) and similar to those in cardiac myocytes. Nevertheless, it was found that arachidonic acid selectively blocks the I_A in smooth muscle cells (Am.J.Physiol.1997). The possibility that the K^+ channel gene for I_A in smooth muscle is deferent from that in cardiac myocytes was examined using RT-PCR techniques. In addition, the related K^+ channels in smooth muscle cells in the rat was cloned. Kv1.4,4.2 and 4.3 have been reported as K^+ channel genes in brain and cardiac myocytes. Based on the expression levels of mRNAs of these genes, it was found taht Kv4.3 is predominant among them in smooth muscle cells. Moreover, Kv4.3 in smooth muscle cells was a new spliced variant of the original Kv4.3 (Kv.4.3M) in brain and has additional 19 amino acids in cytosolic domain close to the C terminus (Kv4.3L) (FEBS Letters, 1997). In the rat heart, the mRNA level of Kv4.3L was higher than that of Kv4.3M.Electrophysiological characteristics of Kv1.4,4.2,4.3M and 4.3L K^+ channels were determined in HEK 293 cells in which one of these channel types was highly expressed. The voltage-dependence of activation and inactivation, the recovery time course from inactivation and the sensitivity to arachidonic acid of Kv4.3L were not significantly different from those of Kv4.3M.Further research is required to elucidate the functional roles of additional 19 amino acids in Kv4.3L.It was also found that the different expression levels of mRNAs encoding the large conductance Ca^<2+> -dependent K^+ channels and the voltage-dependent Ca^<2+> channels in various type of smooth muscle cells, at least in part, explains the difference in membrane excitability in different types of smooth muscle cells.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The activation of muscarinic receptors by exogenously applied acetylcholine (ACh) elicits unique responses in rat iris dilator smooth muscle ; relaxation at low doses and contraction at high doses (1). The pA_2 values of muscarinic antagonists for antagonism to relaxation in dilator muscles were most similar to those for M_3-type muscarinic receptors and contraction might be mediated by M_3-like receptor. The effects of Pertussis toxin (PTX) on contraction and/or relaxation induced by agonists were examined in the rat iris dilator. Only the ACh-induced relaxation was affected by injection of PTX into the anterior eye chamber. Relaxation had completely disappeared after injection of PTX.However, Preteatment with PTX did not significantly affect contraction induced by norepinephrine or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles (2). Therefore, only relaxation of the biphasic response to ACh was mediated by pertussis toxin sensitive GTP binding protein in rat iris dilator. To isolate the gene coding muscarinic receptor expressed in rat iris, RT-PCR was carried out using total RNA of the rat eye as the template and specific primers which were designed based on the sequence of cDNA for the rat cardiac m2 receptor and cDNA for the rat brain m3 receptor.Consequently, PCR products amplified were 1.4k bps for m2 receptor specific primers (3) and 1.8k bps m3 receptor specific primers (4). Each PCR product was subcloned into plasmid vector, and each nucleotide sequence was abalyzed. The protein sequence of m2 receptor cDNA obtained from rat eye differed from that reported for rat cardiac m2 receptor cDNA by 9 amino acid residues. The protein sequence of m3 receptor cDAN obtained from rat eye differed from that reported for the rat brain m3 receptor cDNA by 5 amino acid residues. To confirm the expression of the m2 and m3 receptor molecular subtype around the tissue of rat iris, RT-PCR_Swere performed using specific PCR primers targeted short fragment (350-450 bps) of third intracellular loop. From the results of RT-PCR,it was comfirmed that m2 and m3 receptor mRNAs expressed around the tissue of rat iris. Furthermore, m1 and , m5 receptor mRNA were not expressed substantialy.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : WATANABE Minoru; WALSH Michael; GILES Wayne; UYAMA Yoshiaki; OHYA Susumu; MURAKI Katsuhiko; IMAIZUMI Yuji
     
    The protein responsible for slowly activating outward current upon depolarization (IsK or mini K) in several smooth muscle tissues were cloned using RT-PCR and its distribution was examined using RNase protection assay. The techniques of RT-PCR method were taught in The University of Calgary. The cDNA encoding IsK protein cloned in rat aorta, duodenum, iris, ileum, stomach, trachea and urinary bladder and rabbit aorta, clon, stomach and urinary bladder was 100% identical to that has been reported in the rat kidney. The RNA protection assay showed that IsK is expressed in a similar extent in all these tissues. IsK was recorded upon depolarization in Xenopus oocytes injected with IsK cRNA. Pharmacological experiments using perfusion preparations of the rabbit mesenteric vascular bed suggested that Ba^<2+>-sensitive inward rectifier type K current is partly responsible for the resting membrane potential and tone of mesenteric arterial smooth muscle. In isolated mesenteric arterial myocytes, small K current which is sensitive to Ba^<2+> was observed under whole-cell voltage clamp. Inward rectifier type K channel (IRK1) was cloned from the rabbit mesenteric artery using RT-PCR.Total RNA extracted from mesenteric artery was used as a template. The primers were designed based on the sequence of IRK1 cDNA of the rabbit heart. A PCR product of 1392 bps (RBMAIK1) was obtained from arteries both with or without endothelium. RBMAIK1 was divided into three fragments by restriction endonucleases and subcloned into a plasmid vector. All fragments were, then, sequenced. RBMAIK1 showed 100% identity with rabbit heart IRK1. The injection of cRNA from the RBMAIK1 cDNA into Xenopus oocytes resulted in expression of Ba^<2+> sensitive inward rectifier K current. The techniques of gene transfection to mammalian cell lines will be transferred to Watanabe's group from that in the University of Calgary. Existence of A-type K currents has been reported in several types of smooth muscle cells including portal vein. Kv4.2 or Kv1.4 is the major K channel responsible for A-type K current in the rat heart. To identify the K channels responsible for those in smooth muscle, RT-PCR was performed using the total RNA of several types of smooth muscle tissues as templates and the primers designed from Kv4.2 and 1.4 of the rat brain. The PCR products of about 1.7Kbps were obtained. The subcloning of the PCR products and sequencing will be carried out.
  • イオンチャネル病
  • Molecular Pharmacology of ion channels

Committee Membership

  • 2021/12 - Today   Frontiers in Physiology, Cell Physiology section   Associate Editor
  • 2020/04 - Today   Editor

Other link

researchmap


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.